METHOD AND DICHROGRAPH FOR MEASUREMENT OF CIRCULAR DICHROISM, OPTICAL ROTATION AND ABSORPTION SPECTRA

Abstract: The dichrograph and accompanying method are usable for parallel measurement of circular dichroism according to the first harmonic of photoelectric current of the detector, optical rotation acccording to the second harmonic and transparency according to the null harmonic. It contains the switch (3) of ray, from which alternatively exit two parallel rays - measurement ray (4) and reference ray (5), going through one mutual modulator of elipticity (6), achromatic phase element (9) which is during calibration of circular unit situated in the reference ray (5). Moreover it contains two analyzers - the measurement analyzer (8) in the measurement ray (4) behind the couvette (7) and the reference analyzer (10) in the reference ray (5). The measurement analyzer (8) has the optical axis turned for 45 DEG same as the modulator (6). The reference analyzer (10) is during measurement turned for unit angle, e.g. for 1 DEG , to the measurement analyzer (8).

Description: METHOD AND DICHROGRAPH FOR MEASUREMENT OF CIRCULAR DICHROISM, OPTICAL ROTATION AND ABSORPTION SPECTRA. Technical field- Subject of this invention is the method for measure¬ ment of circular dichroism, optical rotation and absorpti¬ on spectra on optical active, gyrotropic, substancess by spectropolarisation way and construction of an apparatus for performance of this measurement. Background of the invention. Presently circular dichroism spectra can be measured by spectropolarimetric apparatus, dichrographs, which af¬ ter refitting of its optical system can be used for measu¬ rement of other optical properties as optical rotation and/or absorption on tested substance as well. For measu¬ rement of circular dichroism the apparatus have to contain at least the light source of known wav lenght, linear po- larisator, modulator of elipticity, holder and/or couvette for tested substance and detector. For measurement of op¬ tical rotation by the same apparatus there is necessary to install before the detector an analyzer which transfer the polarisation plane into the change of the ray amplitude. For measurement of absorption is advantegous to eliminate, from the optical system, polarisation elements i.e. pola- risator, elipticitry modulator and analyzer. Practically it means that by presently used methods with utilisation of known types of dichrographs all three above mentioned optical properties cannot be measured si- multaneously i.e in real time. It is not convenient mainly in cases when properties or composition of tested substan¬ ces are chaπαinα quickly during the chemical reaction or in the cases when non-stabil organic substances as e.g. hyaiuronidase are measured. The other imperfection of present dichrographs is in fact that they do not enable utilisation of a reference ray for determination of corresponding unit of the circu¬ lar dichroism, because in praxis there are not available etalon substances showing costant circular dichroism in acceptably wide spectral area for example in interval from 300 to 600 nm. This fact limits precision of measurements performed on presently known dichrographs. The goal of invention is to elaborated of the measure¬ ment method which substantially reduces above mentio ned disadvantages and enables, after modification of presently known dichrographs, simultaneous measurement of circular dichroism, optical rotation and absorption spectra in the real time and acceptably wide spectral interval. Disclosure of the invention The above mentioned goal is achieved by the subject of this invention, which is a method for measurement of trans mittance, circular dichroism and optical rotation of opti¬ cal active substance, during which is for determined wave- ienght, after previous calibration of the measurement sys¬ tem and determinati n of unit values assertained by means of harmonic analysis the state of polarisation of measure¬ ment ray with mαduled elipticity after passing through the optically active substance and analyzator, where the chan¬ ges of the measured ray amplitude are transferred onto changes of an electric signals. Subject of the method accor ing to this invention is to assort the null harmonic, the first harmonic and the second harmonic component which correspond to transmi an- ce, circular dichroism and optical rotation of measured optically active substance, while frequention of the first harmonic component corresponds to modulation frequency, and the amplitudes of by this way determinated harmonic components are compared with individual unit values achie¬ ved by calibration. Tha further subject of the method according to this invention with utilisation of the double ray equipment which forms the second ray as a reference ray is, that the reference ray is calibrated and moduled identically with the measurement ray and changes of the amplitude of refe¬ rence ray is transformed on changes of the electric signal changes alternatively with transfering of corresponding changes of measurement ray amplitudes, wheπn the frequen- tion of alternations of both rays, the measurement ray and the reference ray, is at least lOx smaller than the modu¬ lation frequention. Dichrαgraf for providing of this method, formed by the source of the ray of known wavelenght, linear polarisator, modulator of elipticity, connected with the generator of modular voltage, measured sample, analyzer and detector of electromagnetic radiation, to which outlet are connected at least two short range amplifiers according this inven¬ tion has behind the linear polarisator in the inlet ray an direction switch of ray situated, behind which are formed two rays, measurement ray and reference ray, that in the path of the measurement ray is further elipticity modula¬ tor of measurement ray situated, connected to the genera¬ tor of modulation voltage and a measurement analyzer, whe¬ re the optical axes of measurement analyzer is with regard to the optical axis of linear polarisator oriented under the angle 45°, and between them is placed measured sample, for example couvette, and that in the path of reference ray is further elipticity modulator of the reference ray placed, which optical axis is with regard to the optical axis of linear polarisator oriented under the angle 45° and in the path of mentioned reference ray is further at least reference analyzer placed, which optical axes is to the optical axis of linear polarisator oriented under the angle 45°± unit angle o. , and that in paths of the measu¬ rement ray and reference ray are further situated optical elements, advantageously mirrors for directioning of those rays on the mutual detector of electromagnetic radiation, to the outlet of which is connected triad of short range amplifiers, AC amplifier, the first short range amplifier with working frequention corresponding to frequention of elipticity modulators and the second short range amplifier which working frequency corresponds to double frequention of elipticity modulators and that further is in paths of measurement ray and reference ray a ray interruptor situa¬ ted. The unit angle has advantageusly value ±0,1° to ±40°. Moreover may the ray interruptor formt a part of switch of an inlet ray and the elipticity modulator of me¬ asurement ray and the elipticity modulator of reference ray may be formed as mutual elipticity modulator. Finally, according this invention, the source of inlet ray may be formed by monochromator. The measurement method and dichrograph which are sub¬ ject of this invention provide against present stage of technics a substantial progres in the possibility to mea¬ sure simultaneously, in real time, all three above mentio¬ ned optical posessions e.i. circular dichroism, optical rotation and absorption or to test which optical proper- ties can be measuied on actual tested substance. Another advantage of the subject of this invention is possibility ro arrange continual measurement, in wide waveienght scale. Brief description of Drawings Examples of dichrograph arrangements are schematically shown on enclosed drawings. Fig. 1 shows lay out of the dichrograph. Figs. 2 - 4 stages of polarised rays in pla¬ nes A and B according to Fig. 1. Examples of measurement methods and carrying out of the invention. The dichrograph according to Fig 1 is formed by the source 1 of inlet monochromati ray 100 , e.g. laser or monochromatic rays with adjustable waveienght - for exam¬ ple monochromator. The inlet ray 100 goes through linear polarisator 2 and enter the ray swith 3 formed by for example combination of semitransmittable and reflektable mirror. Behing the ray switch 3 the ray 100 is situated an lnterraptor 30 formed for example electronically control¬ led rotation element - chopper. From the interruptor 30 the outlet ray goes alternatively as measurement ray 4 or reference ray 5. The alternation is done by preadjusted switching frekvention. The measurement ray 4 goes further through a modulator 64, of measurement ray elipticity, measured sample 7, e.g. couvette with the solution, parameters of which are to be measured, measurement analyser 8, which is formed by for example cryslall polarisator of common construction and mirror 11 to a detector 13 which can be some sensor of electromagnetic radiation for example some convenient pho¬ toelectric censor as photoelectric multiplier The reference ray 5 goes through the reference modula¬ tor 65 of elipticity which is the same as the measurement modulator 64 of measurement ray. Advantageously both modu¬ lators can be realised as a mutual modulator 6. Then the reference ray 5 goes through achromatic quarterwave phase element 9 which can be formed by couple of mutually coun¬ ter oriented plates made from anizotrop material. One of those plates is radially movable and into the reference ray 5 is positioned alternatively as mentioned further. The reference ray 5 goes further through the refrence ana¬ lyzer 10, which has the same possessions and parameters as the measurement analyzer 8. After exit from the reference analyzer 5 is, same as the measurement ray 4, directed by means of the mirror 12 on the same detector 13 of electro¬ magnetic radiation. On outlets 134, 135 and 136 of detector 13 triad am¬ plifiers, AC amplifier 14, the first short range amplifier 15 and the second short range amplifier 16. Short range amplifiers 15 a 16 are formed for example as synchronous detectors in common connection and are connected with ge¬ nerator 17 of alternate voltage from which one voltage of lower frequention is used for control of modulator 64 and 65 of elipticity of measurement and reference ray or mutu¬ al regulator 6 of elipticity. The second voltage has dou¬ ble frequention and is used for the second short range am¬ plifier 16. One of necessary condition for proper function of the dichrograph constructed according to this invention is that the working frequention of generator 17 is sub¬ stantially, at least 10 times larger, than the working frequency of the ray interuptor 30 i.e. frequency of swit¬ ched inlet iay 100 into direction of measurement ray 44 and reference ray 5. On execution ace. to the Fig. 1 the working frequention of the interaptor 30 of rays for exam¬ ple 100 Hz, while the working frequention of generator 17 of modulation voltage is 50 kHz. The whole system of dichrograph according to Fig. 1 is completed by control, registration and evaluation equi¬ pment 18 vhich are not binded on the subject of this in¬ vention and can consist of the control computer equipped by necessary software. On E'ig. 2 are screened, to the plain of drawing, space stages of polarisation of the inlet ray 100 in the plane A according to the Fig. 1 - i.e. in front of the measured sample 7. Corresponding electric vector 41 is transferred during modulation into the form of ellipsis, ring and vice versa. The linearly polarised state corresponds to the state of polarisation under zero voltage on the modulator 64 of the measurement ray or on modulator 65 of the refe¬ rence ray 5. Fig. 3 shows position of electric vector 41 of linear¬ ly polarised inlet ray 40 in the plane B according to the Fig.l i.e. after its passing through the couvette 7 with measure sample in the case that this shows the optical ro¬ tation only. Fig. No.4 shows positions and/or states of the electric vector 41 in the case that the measured sam¬ ple shows circular dichroism only. It is seen from the Fig. No. 4 that amplitudes of the leftturning component 421 and rightturning component 422 are due physical pro¬ perties of measured substance different. Result of of this fact is that after their interferention on the outlet from the measured sample 7 the end of corresponding resulting electric vector forms the ellipsis, which is market on the Fig.4 dashed. In the case that the measured sample 7 shows as well optical rotation as circular dichroism the axis of resulting ellipsis is turned for certain angle >-fi and el- lipticity is determined by the angle "ψ which tangenta is determined by ratio of the small halfaxis 442 and large halfaxis 441 as is seen on the Fig. 4. Due to the fact that the measured sample 7 shows practically always some absorption the amplitude of corresponding vector on the plane B according to Fig.l, i.e. behing the measured sam¬ ple 7 is smaller than corresponding amplitude of the sam vector on the plane A i.e. before the measured sample 7. Measurement of optical parameters values is based on comparison of measurement ray 4 state before the measure¬ ment analyzer and reference ray 5 before the reference ana lyzer 10 according the Fig.l. The measurement ray 4 is for example after passing by modulator 64 of measurement ray 4 elipticity modulated so as shown on the Fig.2. In the case that the measured sam¬ ple 7 shows all three measured parameters i.e. some absor¬ ption, circular dichroism and optical rotation, amplitudes as well as corresponding intensities of the ray electric components are changing proportionally to values of measu¬ red parameters. Those changes are related to the original plane 400 of linearly polarised measurement ray and cor¬ responding angle position of the measurement analyzer 8. In the second wing, i.e. in a reference ray 5, which is polarised on the outlet from the modulator 65 of the reference ray 5 same as the measurement ray 4 on the out¬ let from the modulator 64. The reference analyser 10 is according this invention oriented so, that its polarisati¬ on plane is with regard to corresponding polarisation pla¬ ne of measurement analyzer 8 anglely turned for certain preselected and adjusted unite angle o. , for example 1°. After passing of the reference ray 5 through reference analyser 10 intensities I of the measurement ray 4 and re¬ ference ray 5 are changed so that on outlets 134, 135, 136 of detector 13 are generated electric currents which con¬ tain zero harmonic component proportionate to the absorp¬ tion of measured substance, the first harmonic component proportionate to the value of circular dichroism of measu- red substance and the second harmonic component proportio¬ nate to optical rotation of measured substance. Individual measured components are related to the basic harmonic which corresponds to the generator 17 frequency of modula¬ tion voltage of modulator 64 and 65 of measuring and refe¬ rence rays. For amplitudes of individual specified harmo¬ nic components following relations are valid" for the null harmonic Io = o x T for the first harmonic Ii = ki x sin ^ for the second harmonic I2 = 2 x sin* where - 1 = transmissivity coefficient of measured substance = </t «rc,s ,n _ _- -ft. + i t, . R. - L - transmittance coefficients of circu¬ larly polarised rays R and L a R. O m. - phase shifting of rays R and L if/ - elipticity caused by circular dichroism ψ - turning of the polarisation plane caused by the optical rotation Coefficients ko . ki and k2 are determined as constans of a measurement system by calibration before the measure¬ ment as follows' Coefficient ko is determined by difference of photoe¬ lectric currents in cases when the measured sample 7 is formed by the couvette accordinghe Fig. 1 filled by the substance with transmi tance ( = 1, e. g. dest.il led water a than by substance with transmittance T ~" = 0, what can be simulated by closing of measurement ray 4. For measurement of coef icient ki is into the measure¬ ment ray 4 installed, before measurement analyzer 8, an achromatic quarterwave phase elemen 9 oriented under the angle 45° with reference to the plane of electric vector 40 which together with the measurement analyzer 8 oriented with reference to the plane of electric vector 40 under the angle 45°+ " . simulates presence of etaIon achromatic dichroic substance, which causes elipticity of the measu¬ rement ray 4 of value 1" ~ -<■*■ The value ' is independent on the waveienght of source 1 of inlet ray 100. The value ki is determined from the difference of values photoelect¬ ric currents under orientation of the measurement analyzer 8 eider under the angle 45°,or after its following turning for selected unit angle e.g. 1°. In praxis it is advanta¬ geous to select the unit angle from the interval ± 0,1° to ± 40°. Similarly is after elimination of the achromatic quarterwave phase element 9 determined coefficient k2 - By the same way it is possible to measure proportiona¬ lity coefficients ko , ki and k2 corresponding to the refe¬ rence ray 5. All coefficients of proportionality are re¬ gistered in the evaluation equipment 18. During the measu¬ rement of a few wavelenghts for example with monochromator as the source 1 of inlet monochromatic ray 100 is determi¬ nation of coefficients ko. ki and k done analogically for all demanded wavelanghts. After completing of calibration which is done after the first installation and/or for periodical testing mea¬ surements the achrommatic quarterwave phase element 9 is eliminated from the optical path and the reference analy¬ zer 10 remains turned for mentioned unit angle. The measu- * rement analyzer 8 is turned into the original position 0* ~* 0. During the measurement of the sample 7 ire due to the interruptor 30 of the ray compared amplitudes of null har- manic, measurement and reference rays, what enables to achieve the transmittance coefficient. of measured sam¬ ple 7 and in the same time amplitudes of the irst and se¬ cond harmonic corresponding to the measurement ray 4. are compared with the unit amplitudes of the second harmonic which corresponds to the reference ray 5, what enables to receive values of circular dichroism and optical rotation. Ratio of the first and second harmonic of the reference ray 5 was obtained during calibration and is memorised and registered during calibration by the evaluation equipment 18. Utilisation of the reference ray 5 which is advantage- nously modulated for example by common modulator 6 by the same way as the measurement ray 4 enable to achieve high reproducebi 1 ity of measurement of absorption, circular dichroism and optical rotation due to elimination of fluc¬ tuation of the source 1, mutual modulator 6, elipticity, sensitivity of detector 13 and by this fluctuation of the whole connected electronic chain. The stability of mutual modulator 6 is moreover during measurement continually as¬ sured by continual check up of reference ray 5 ratio of null and second harmonic amplitudes. The high precision of measurement is given by the preci¬ sion of micrometric screws for turning of the measurement analyzer 8 and reference analyzer 10. Increase of circular dichroism measurement precision is achieved by simulation of an achromatic unit etalon by combination of the achro¬ matic quarterwave phase element 9 and the reference analy¬ ser 101 which can be under presence of achromatic quarter- wave phase element 9 utilised as a compensator of circular dichroism. In this case the dichrograph enables measure¬ ments with the maximum precision by means of null compen¬ sation method. The method for measurement of spectropolaπmetric pos¬ sessions of optically active substancess and the dichrog¬ raph according to this invention can be, thanks to possi¬ bility to have immediately available values of absorption, optical rotation and circular dichroism and to possibility to realise measurement for wavelenghts selected from the determined spectral range, successfully utilised for rese¬ arch of gyrotropic substances namely fron area of organic chemistry, farmacology, biophysics, biochemistry etc. List of reference characters. Source 00 Inlet ray Linear polarisator 0 Plane Cof linearly polarised waving) Switch of ray 0 Ray interruptor Measurement ray 0 Inlet ray of modulator 00 Plane Cof linearly polarised ray) 1 Electric vector Cof linearly polarised ray) 2 Electric vector Cof circularly polarised ray) 20 Addition electric vector 21 Left turning component 22 Right turning component 3 Electric vector Cof eliptically polarised ray) 4 Resulting electric vector 41 Great halfaxis 42 Small halfaxis 43 Axes of ellipsis Reference ray Mutual elipticity modulator 0 Optical axis of modulator 4 Elipticity modulator of measurement ray 5 Elipticity modulator of reference ray Measured sample couvette) Measurement analyzer 80 Optical axis of nesureraent analyzer 9 Achromatic quarterwave phase element 90 Optical axis of achromatic quarterwave phase element 10 Reference analyzer 101 Optical axis of analyzer 11.12 Mirror 13 Detector 134 The first output of detector 135 The second output of detector 136 The third output of detector 14 AC amplifier 15 The first short range amplifier 16 The second short range amplifier Generator of modular voltage Control, registration and evaluation equipment I Ray intensi y ko.k1.k2 Harmonic components proportionality coefficients o The unit angle of the analyzer turning Ccircular dichroism) I 6 The unit angle Cof the analyser turning - optical rotation) "~ ~ Measured sample trans ittation coefficient f Elipticity caused by circular dichroism f Plane polarisation turning angel Ccaused by the optical rotation) OR. d im Phase shifting of R and L circular component of rays R. L Right and left circular component of the ray

Claims: L A I M S 1) A method for measurement of transmittance. circular dichroism and optical rotation of optical active substan¬ ce, during which is for determined waveienght. after pre¬ vious calibration of the measurement system and determina¬ tion of unit values assertained by means of harmonic ana¬ lysis the state of polarisation of measurement ray with moduled elipticity after passing through the optically ac¬ tive substance and analyzator, where the changes of the measured ray amplitude are transferred onto changes of an electric signals, characterized by assorting the null har¬ monic, the first harmonic and the second harmonic compo¬ nent which correspond to transmittance, circular dichroism and optical rotation of measured optically active substan¬ ce, while frequention of the first harmonic component cor¬ responds to modulation frequency, and the amplitudes of by this way determinated harmonic components are compared with individual unit values achieved by calibration. 2) A method of measurement according to claim 1 with utilisation of the double ray equipment which forms the second ray as a reference ray, characterized in that, that the reference ray C5) is calibrated and moduled identical¬ ly with the measurement ray C4) and changes of the ampli¬ tude of reference ray C5) is transformed on changes of the electric signal changes alternatively with transfering of corresponding changes of measurement ray C4) amplitudes, vherin the frequention of alternations of both rays, the measurement ray C4) and the reference ray C5), is at least lOx smaller than the modulation frequention-. 3) The dichrograph for providing of the method accor¬ ding to claim 2 formed by the source of the ray of known waveienght, linear polarisator, modulator of elipticity, connected with the generator of modular voltage, measured sample, analyzer and detector of electromagnetic radiati¬ on, to which outlet are connected at least two short range amplifiers characterized in that, that behind the linear polarisator C2) is in the inlet ray C100) an direction switch C3) of ray situated, behind which are formed two rays, measurement ray C4) and reference ray C5), that in the path of the measurement ray C4) is further elipticity modulator C64) of measurement ray C4) situated, connected to the generator C17) of modulation voltage and a measure¬ ment analyzer C8). where the optical axes C80) of measure¬ ment analyzer is with regard to the optical axis C20) of linear polarisator C2) oriented under the angle 45°. and between them is placed measured sample C7), for example couvette. and that in the path of reference ray C5) is further elipticity modulator C65) of the reference ray C5) placed, which optical axis C60) is with regard to the optical axis 20) of linear polarisator C2) oriented under the angle 45° and in the path of mentioned reference ray C5) is further at least reference analyzer CIO) placed, which optical axes C101) is to the optical axis C20) of linear polarisator C2) oriented under the angle 45° ±unit angle OO, and that in paths of the measurement ray C4) and reference ray C5) are further situated optical elements, advantageously mirrors 11.12) for directioning of those rays on the mutual detector 13) of electromagne¬ tic radiation. to the outlet of which is connected triad of short range amplifiers, AC amplifier C14), the first short range amplifier C15) with working frequention cor¬ responding to frequention of elipticity modulators C64.65) and the second short range amplifier C16) which working frequency corresponds to double frequention of elipticity modulators C64.65) and that further is in paths of measurement ray C4) and reference ray C5) a ray inter¬ ruptor C30) situated. 4) The dichrograph according to claim 3, characterized in that, that the unit angle C ex.) has value ±0,1° to ±40°. 5) The dichrograph according to claim 3 or 4, charac¬ terized in that, that the ray interruptor C30) forms a part of switch C3) of an inlet ray C100). 6) The dichrograph according to some of claims 3 to 5 characterized in that, that the elipticity modulator C64) of measurement ray C4) and the elipticity modulator C65) of reference ray C65) are formed as mutual elipticity modulator C6) . 7) The dichrograph according to some of claims 3 to 6. charakterized in that, that the source CD of inlet ray C100) is formed by monochromator.

Inventors: ROKOS JIRI, ROKOS, JIRI
Applicants: ROKOS & CO LTD, ROKOS JIRI, ROKOS & CO, LTD., ROKOS, JIRI
Application year: 1994
Main classification: G01N21
IPC codes: G01N21